Pseudomonas aeruginosa (PA) is one of the most virulent opportunistic pathogens of man. The morbidity of PA infections results from the ability of the bacterium to colonize previously injured or disrupted epithelial cell layers leading to further epithelial cell damage, inhibition of wound healing, and access to other tissues or the blood stream. Our initial work utilized a novel genetic screen to identify new virulence factors of PA required for epithelial cell injury. These studies identified new virulence factors (the type III secretion system and the secreted effector protein ExoU) and also suggested new functions in virulence for previously identified virulence factors (type IV pill). The role of these genes in pathogenesis was validated using assays testing for virulence in the tissue culture system and in a mouse model of acute pneumonia. We discovered that PA can damage epithelial cells and macrophages by at least two type Ill-secretion dependent pathways. The first involves ExoU-mediated necrosis and the second pathway has features of apoptosis. In this competitive renewal we will continue these studies with the long term goals of (i) understanding the complex interplay between the bacterial type III secretion system, its secreted effectors, and the host eukaryotic cell and (ii) elucidating the role of type IV fimbriae as virulence factors in acute infections caused by PA. Our short term goals will focus on (i) the pathways by which ExoT alters the host cell cytoskeleton, (ii) the mechanism of type III secretion-dependent apoptosis, (iii) and the role of type IV pill in type III secreetion. Specific aim 1. We will test the hypothesis that multiple domains of ExoT contribute to its role in inhibiting bacterial internalization, inducing cytoskeletal changes and cell rounding, and inhibiting wound healing of eukaryotic cells. Specific Aim 2. We will dissect the mechanism by which type III secretion induces apoptotic like death in host cells. Specific Aim 3. We will explore the biological roles of the polarly located type IV pill in virulence. We will test the hypothesis that specific components of type IV pill are required for discrete steps in type Ill-mediated secretion and translocation.